Metal-Enhanced Fluorescence of Conjugated Polyelectrolytes with

Mar 10, 2011 - Conjugated polymers (CPs) have attracted great research interests in ... Ag NPs density of platform on PFVP fluorescence enhance- ment...
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ARTICLE pubs.acs.org/JPCB

Metal-Enhanced Fluorescence of Conjugated Polyelectrolytes with Self-Assembled Silver Nanoparticle Platforms Junlong Geng,† Jing Liang,† Yusong Wang,† Gagik G. Gurzadyan,‡ and Bin Liu*,† †

Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of Singapore, Singapore 117567, Singapore ‡ Division of Physics and Applied Physics, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore 637371, Singapore

bS Supporting Information ABSTRACT: We report the use of a simple Ag nanoparticle (NP) platform to enhance the fluorescence signatures of conjugated polyelectrolytes (CPEs). Ag NP platforms with different extinction intensities were fabricated by self-assembly of Ag NPs on NH3þfunctionalized glass surface. Layer-by-layer (LBL) deposition of oppositely charged polyelectrolytes is used to control the Ag NPCPE distance. The Ag NP platforms with high optical densities provide higher fluorescence enhancement factors for CPEs as compared to those with low optical densities. In addition, the CPE fluorescence enhancement is found to be directly related to the overlap between the absorption spectra of CPEs and the extinction spectra of Ag NP platforms. Both steady-state and time-resolved fluorescence spectroscopic studies reveal that the fluorescence enhancement is controlled by the increase in both absorption and radiative decay rates of the CPEs in proximity of Ag NPs. The enhanced CPE fluorescence signature is further used to detect single-stranded DNA using a Cy5 dye labeled peptide nucleic acid probe (Cy5-PNA) through F€orster resonance energy transfer (FRET). We anticipate that the organic-inorganic hybrid platform will provide new opportunities for CPE application in sensing and device fabrication.

’ INTRODUCTION Conjugated polymers (CPs) have attracted great research interests in light-emitting diodes,1,2 field-effect transistors3,4 and chemo/biosensors,5,6 due to their delocalized π-conjugated backbone, high-conductivity, high absorption coefficient and light-harvesting property. Conjugated polyelectrolytes (CPEs) are CPs with pendant ionic functionalities. The charged nature and solubility in polar solvents provide additional advantages for CPEs to be used in chemo/biosensor7,8 and optoelectronic devices.9,10 It has been generally observed that highly fluorescent CPEs lead to high detection sensitivity11,12 and good device performance.13,14 The strategies to improve CPE fluorescence usually involve polymer backbone15 or side-chain16 modification which is tedious, and more importantly not generic. Fluorescence enhancement of fluorophores by metal nanostructures is an active area of research.17-20 Metal-enhanced fluorescence (MEF) arises from the near-field interaction between fluorophores and metal nanostructures, most typically of Ag and Au. Two factors have been generally considered for MEF of fluorophores. When fluorophores are in close proximity to metal nanostructures, they are exposed to increased electric fields around the nanostructures, resulting in significant increases in their absorption cross sections.21-23 This can lead to a subsequent increase in the excitation and eventually enhancement in fluorophore fluorescence. Another factor is plasmon effect, which r 2011 American Chemical Society

ascribes the enhancement to the interaction between fluorophore excited state and induced surface plasmon of metal structures.24,25 An excited-state fluorophore can induce a nearby metal structure to create plasmons. The induced plasmons radiate an emission which shows the photophysical characteristics of the coupling fluorophores. The radiative decay rates and the quantum yields for fluorophores can increase in vicinity of metal structures, in contrast to the “naked” fluorophore itself.20 Most studies on MEF are focused on the investigation of factors that influence the magnitude of fluorescence enhancement for the same fluorophore by different metal nanostructure architectures.17-20 MEF has been widely utilized to enhance fluorescence of small organic dyes,26,27 and quantum dots.28-30 However, the application of MEF to CPs,31,32 especially CPEs,33 has been rarely reported. In the previous study, we have shown that Ag NPs are able to enhance the fluorescence of a yellow emission CPE,33 however, the factors that affect CPE fluorescence and the detailed mechanism for CPE fluorescence enhancement have not been explored. In this contribution, we report a detailed study of various factors that affect the CPE fluorescence on Ag NP platforms. We Received: September 28, 2010 Revised: February 18, 2011 Published: March 10, 2011 3281

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The Journal of Physical Chemistry B start with the construction of CPE-Ag NP platforms with layerby-layer (LBL) technique to control the distance between Ag NPs and CPEs. Taking poly[9,90 -bis(6-N,N,N-trimethylammonium) hexyl)-2,7-fluorenyldivinylene-alt-1,4-phenylene dibromide] (PFVP) as a representative CPE, we investigate the influence of the distance between Ag NPs and PFVP and the Ag NPs density of platform on PFVP fluorescence enhancement. We further explore the excitation-wavelength dependent PFVP fluorescence amplification. In addition, the effect of spectral overlap between the extinction of Ag NP platforms and the absorption of CPEs on the enhancement factor for different CPEs is also studied. At last, as CPEs have been widely utilized in FRET based biosensors to improve detection sensitivity,7,8 the enhanced fluorescence signal of PFVP by Ag NP platforms is further used to detect single-stranded DNA (ssDNA) using Cy5-labeled peptide nucleic acid probe on surface.

’ EXPERIMENTAL SECTION Materials. Poly[2,7-(9,90 -bis(6-N,N,N-trimethylammonium)

hexyl)fluorene-co-1,4-phenylene dibromide] (PFP), poly[9,90 bis(6-N,N,N-trimethylammonium)hexyl)-2,7-fluorenyldi vinylene-alt-1,4-phenylene dibromide] (PFVP), poly[9,9-bis(60 -N, N ,N-trimethylammonium)hexyl)fluorene-alt-4,7-(2,1,3-benzothiadiazole) dibromide] (PFBT), and poly[9,9-bis(60 -N,N,N-trimethylammonium)hexyl)fluorenyldivinylene-alt-4,7-(2,1,3,-benzothiadiazole) dibromide] (PFVBT) were synthesized according to the previous reports.34-37 DNA oligonucleotides were purchased from IDT including the cDNA (ssDNAc): 50 -TGA GAT GCC GTG GA-30 and noncDNA (ssDNAnc): 50 -GGT CAT TAG CTT CT-30 . The Cy5-labeled PNA (Cy5-PNA) has the sequence of 50 -Cy5-OO-TCC ACG GCA TCT CA-Lys-Lys-30 , which was ordered from Panagene, Korea. Poly(diallyldimethylammonium chloride) (PDDA; MW 200 000-350 000, 20 wt % in water, Aldrich), poly(styrene sulfuric acid) sodium salt (PSS; MW 70000, Alfa-Aesar), 1-methyl-2-pyrrolidinone (NMP) (99.3þ%, Biotech grade, Sigma-Aldrich), silver nitrate (Sigma-Aldrich, reagent plus, >99.8%), sodium citrate tribasic dehydrate (Sigma, ACS regent, g99.0%), sodium borohydride (g99%, Sigma), sodium chloride (g99.5%, Sigma), 3-aminopropyl-triethoxysilane (APTES, 99%, Aldrich), sulfuric acid (98.0%, BDH), acetic acid (100%, Merck), methanol (99.8%, Merck), ethanol (99.9%, Merck), hydrogen peroxide (30%, Merch), ammonia solution (30 wt %, SINO Chemical Company), phosphate buffered saline (PBS, 1500 mM), transparent waterproof tape, glass microscope slide (VFM microscope slides, 26  76 mm, 1.0-1.2 mm; CellPath), and silicon wafer (Φ 101 ( 0.5 mm) were used as received. Milli-Q water (18.2 MΩ) was used to prepare all solutions. Methods. Synthesis of Silver NPs in Solution. The silver NPs were synthesized using a seed-mediated growth method.38 All glassware and stir bars used in the synthesis were thoroughly cleaned in aqua regia (HCl:HNO3 = 3:1), rinsed with Milli-Q water and dried prior to use. First, ∼5 nm Ag NP seeds were prepared by chemical reduction of silver nitrate with sodium citrate and sodium borohydride solution. One mL of 20 mM silver nitrate solution and 0.8 mL of 80 mM sodium citrate solution were added into 17.8 mL of Milli-Q water and refluxed for 1 min. 400 μL of 0.1 mM sodium borohydride solution was added dropwise into the above solution, and refluxed for 5 min. The size of Ag NPs was increased by the seed-mediated growth

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method. Typically, 8 mL of as-synthesized Ag seed solution (1 mM) and 0.2 mL of 80 mM sodium citrate solution were added to 38 mL of Milli-Q water. The mixture was heated to 100 °C under reflux. 2.1 mL of 20 mM AgNO3 solution was added dropwise in 5 batches, with 10 min between each addition. The solution mixture was heated for another 20 min before it was cooled down to room temperature. The final Ag NPs were obtained by repeating the above seed-mediated growth procedure two more times. The obtained Ag NP suspension has a final concentration of 0.2 nM. Field emission scanning electron microscopy (FE-SEM) showed that the obtained Ag NPs have an average diameter of ∼60 nm. NH3þ-Functionalization of Glass Substrates. The standard 75  25 mm microscope glass slides were cut into ∼12.5 mm 7.5 mm dimension and washed with water. The slides were then treated in piranha solution (H2O2:H2SO4 = 1:3) at 80 °C for 30 min, followed by sonication with RCA solution (H2O:H2O2: NH3 = 5:1:1 V/V/V) for 30 min. Next, the slides were rinsed with Milli-Q water, ethanol, Milli-Q water sequentially and finally dried with nitrogen gas. The cleaned glass slides were silanized by incubating in a silane solution (2% APTES in a mixture which contains methanol, Milli-Q water and acetic acid, V:V:V = 95:5:0.1) with gentle shaking for 30 min. The silanized glass slides were rinsed with ethanol, sonicated in ethanol for 2 min, and stored in ethanol for further use. Self-Assembly of Ag NPs on Glass Substrates. Prior to Ag NP assembly, one side of silanized slides was covered with a waterresistance transparent adhesive tape and the slide was then immersed in the Ag colloid solution for self-assembly. To prepare Ag NP platforms with different densities, the glass slides were immersed in Ag colloid solution (80 μL of stock silver solution and 520 μL of Milli-Q water) for 2 to 12 h. The self-assembled Ag NP platforms were then rinsed with Milli-Q water, and stored in 800 μL of Milli-Q water in a 24-well microplate for UV/PL measurement using a Tecan Microplate Reader. Self-Assembly of CPEs onto Ag NP Platform Surface. LBL assembly of PSS/PDDA on NH3þ-functionalized glasses or Ag NP platform was carried out manually.39 To form Ag NP platforms, the substrates were immersed in 800 μL of PDDA solution ([repeat unit] = 10 mM in 0.1 M NaCl aqueous solution) for 15 min, rinsed with water, and immersed in PSS solution ([repeat unit] = 10 mM in 0.1 M NaCl aqueous solution) for 15 min, followed by washing with water. Multilayer assemblies consisting of alternating layers of PDDA and PSS were prepared by consecutive adsorption on the top of multilayer assemblies based on NH3þ-functionalized glass slides or Ag NP platform slides. Finally, the above slides were incubated in CPE solutions ([repeat unit] = 2.5 μM in 800 μL water) for 5 min. After washing with Milli-Q water, the prepared slides were stored in 800 μL of water in a 24-well microplate for UV/PL measurement. DNA Assay on CPE-Ag NP Platforms. For DNA detection, Cy5-PNA and complementary ssDNAc or noncomplementary ssDNAnc were first mixed together in the incubation buffer (25 mM PBS) to form a duplex structure. These mixed solutions were then heated up to 80 °C for 10 min, followed by natural cooling to room temperature. The annealed Cy5-PNA/ssDNAc or Cy5-PNA/ssDNAnc mixture was diluted with incubation buffer to 300 μL and yielded a concentration of 10-7 M. The obtained solutions were incubated with PFVP assembled on Ag NP platform substrate or on plain glass substrate for 15 min, respectively. After washing with incubation buffer, the treated 3282

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Scheme 1. Schematic Illustration of the CPE-Ag NP Platform Construction and the Chemical Structures of PFP, PFVP, PFBT, and PFVBT

slides were placed into 800 μL water in a 24-well microplate for UV/PL measurement by Tecan MicroplateReader. Characterization and Instrumentation. The extinction spectra of Ag NPs were measured using a Shimadzu UV-1700 spectrophotometer. The prepared Ag NPs were characterized by FESEM on a JEOL JSM-6700F operating at 5 kV. The assembled Ag NPs on glass slides were imaged by atomic force microscope (AFM) (Nanoscope III, Digital Instruments) operating in the tapping mode with a silicon tip. The thickness of polymer films was measured on the surface of a similarly prepared silicon wafer by using a spectroscopic ellipsometer (VB-250, VASE Ellipsometer, J. A. Woollam Co., Inc.) after drying the samples. The wavelength ranging from 500 to 1000 nm and the angles of incident light (65° and 75°) were used for the spectroscopic ellipsometer, which was equipped with a 75 W light source and a high-speed monochromator (HS190, J.A. Woollam Co., Inc.). For each sample, three different locations were measured and the thickness was obtained by using softwares provided by the manufacturer. The absorbance change of PFVP solution before and after slide assembly was monitored by MicroplateReader (TECAN Infinite M200). A similar monitoring process was done to monitor other CPE solutions before and after assembly. All sample slides were placed at the bottom of a 24-well microplate for measurement of the extinction spectra or steady-state fluorescence spectra using a Microplate Reader (TECAN) operated in top reading mode. All spectra of sample slides were measured in Milli-Q water and corrected after subtraction of background. Fluorescence lifetime measurements were performed on a FluoTime 200 TCSPC fluorescence platform from Picoquant GmbH (Berlin, Germany). A Titanium-sapphire 100 fs laser (Chameleon, Coherent) with second- and third-harmonic generation was used as the excitation source, and it is excitation wavelength was 390 nm. In time correlated single photon counting (TCSPC) apparatus, the detector is based on a microchannel plate (MCP) PMT system HAM-R3809U-50 (Hamamatsu) and has a spectral sensitivity from 160-850 nm and instrument response function of 30 ps. Fluorescence lifetimes were extracted from the decay curves using commercially available fluorescence lifetime analysis software (FluoFit Pro. PicoQuant GmbH). Fluorescence decay curves were fitted using a two or three exponential mode.

’ RESULTS AND DISCUSSION Fabrication of CPE-Ag NP Platforms. Scheme 1 shows the MEF platform design and CPE (PFP, PFVP, PFBT, PFVBT) structures. The platform preparation started with self-assembly of negatively charged Ag NPs onto NH3þ-functionalized glass slides. Poly(diallyldimethylammonium chloride) (PDDA) and poly(styrenesulfonic acid) sodium salt (PSS) were subsequently self-assembled to control the distance between the Ag NPs and the CPE layer. In the last step, cationic CPEs were assembled onto negatively charged PSS surface as the top layer. Figure 1 shows absorption and emission spectra of the selected polymers. PFP, PFVP, PFBT and PFVBT have absorption maxima at 387, 430, 449, and 530 nm, and their corresponding emission maxima are 415, 470, 582, and 639 nm, respectively. The significant difference in the absorption spectra among different CPEs allows us to study the effect of spectral overlap between CPE absorption spectrum and Ag extinction spectrum on fluorescence enhancement. The citrate-protected Ag NPs were synthesized using a seedmediated growth method.38 The FE-SEM image shows that Ag NPs have an average size of ∼60 nm (Figure 2A). The carried negative charges of Ag NPs due to absorbed citrate molecules allowed them binding onto NH3þ-functionalized glass slides. With increased assembly time, the color of Ag NP platforms changed from light yellow to yellow by visual inspection, which indicated that more Ag NPs were deposited onto the surface of glass slides. A quantitative analysis of the films was obtained by measuring the extinction spectra of these films (Figure 2B). Slides prepared with Ag NPs at 2 h exposure time have shown a characteristic extinction peak centered at ∼440 nm. With increased exposure time from 2 to 12 h, the intensity of the extinction peak increases, which is accompanied by a blue-shift in spectral maximum from ∼440 to ∼420 nm.40 The observed blue shifts of Ag NP platform indicate that more electron delocalization among Ag NPs with increased deposition time. To obtain a more detailed understanding of the surface for Ag NP platforms, the surface topographies for platforms with extinction intensities of 0.22 (4 h), 0.40 (8 h), and 0.61 (12 h) were studied by AFM. The AFM images shown in Figure 3 clearly indicate that the elongation of silver deposition time from 2 to 12 h indeed increases the Ag NP density on the glass surface 3283

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Figure 1. (A) Normalized UV-vis absorption spectra of PFP, PFVP, PFBT, and PFVBT in films. (B) Normalized emission spectra of PFP, PFVP, PFBT, and PFVBT in films upon excitation at 387, 430, 449, and 530 nm, respectively.

Figure 2. (A) FE-SEM of the Ag NPs used for platform preparation. (B) The extinction spectra of Ag NP platforms prepared with different Ag selfassembly time (2-12 h).

Figure 3. AFM images of Ag NP platforms with different extinction intensities: (A) I = 0.22, (B) I = 0.40, and (C) I = 0.61.

with shortened interparticle distance. The Ag NP density of the film with 12 h deposition time (Figure 3C) has 38 NP/μm2, which is significantly improved as compared to that with 2 h deposition time (∼16 NP/μm2, Figure 3A). These date clearly indicate that particle density and interparticle distance on the platform can be controlled by simply changing the deposition time. Influence of CPE-Ag NP Distance on CPE Emission. As the fluorescence enhancement on Ag NP surface is reported to be strongly distance dependent,39,41-45 we first investigated the

fluorescence enhancement of CPEs as a function of CPE-Ag NP distance. LBL technique, a versatile and powerful method to grow thin polymeric films on solid substrates based on electrostatic interaction between oppositely charged polyelectrolytes,39 is used to fine-tune the Ag NP-CPE distance. Here, the Ag NP platform with extinction intensity of ∼0.6 was chosen, and PFVP was used as a representative CPE as its absorption has the best overlap with the extinction spectrum of Ag NPs platform21 (Figure S1 in the Supporting Information). A set of experiments were conducted to monitor PFVP fluorescence atop of PDDA/ 3284

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Figure 4. (A) Distance dependent emission spectra of PFVP on the reference glass slide and Ag NP platform (I = 0.61) as a function of spacer (PDDA/ PSS)n (n = 1-4) bilayers, excited at 430 nm. (B) Fluorescence decay of PFVP on the reference glass slide and Ag NP platform as a function of spacer (PDDA/PSS)n (n = 1-4) bilayers. Instrument response function (IRF) is also indicated.

PSS layers assembled on both Ag NP platforms and glass reference slides. The average thickness of each bilayer is estimated by elipsometry, to be ∼3 nm which is in accordance with previous reports for deposition PDDA/PSS on polystyrene particle surface.39 The PFVP-Ag NP distance is thus varied from ∼3 nm to ∼12 nm as one bilayer to four bilayers of PDDA/ PSS are built between them. Figure 4A shows the fluorescence spectra from PFVP-adsorbed PDDA/PSS layers with varied distances from the Ag NP platform as well as that for the same amount of PFVP adsorbed on glass reference slide. The polymer fluorescence reaches a maximum at two bilayers (∼6 nm), and an overall maximum enhancement factor (the ratio of fluorescence on Ag NP platform to that on reference) of 6.5 is observed. The PL intensity decreases when the spacer is further increased to three and four bilayers. However, as shown in Figure S2, Supporting Information, there is no obvious difference in PFVP emission intensity as a function of distance between PFVP on the reference glass substrates. A similar trend is also observed for PFP, PFBT and PFVBT (Figures S3-S5 in the Supporting Information). In order to understand the origin of fluorescence enhancement, the lifetimes for PFVP on Ag NP platforms with different distances as well as that on glass slides were measured, and the corresponding fluorescence decay curves are shown in Figure 4B. Deconvolution/fit of decay curves results in three exponents with major contribution (>95%) of 50-200 ps components. It is obvious that PFVP on the Ag NP platform shows faster intensity decay rates as compared to that on the glass substrate. The amplitude weighted average lifetime of PFVP on the Ag NP platform with a spacer of two-bilayer is 0.11 ns compared to 0.22 ns on glass slides. The shortening of lifetime and the observed fluorescence intensity on the Ag NP platform are due to the interaction of excited-state PFVP with plasmonic Ag NPs, in agreement with the previous reports.24,25,35 In addition, the average lifetime slightly increases with increased distance from the Ag NP surface, and the amplitude weighted average lifetimes for PFVP on the Ag NP platform with one, three, and four bilayer-spacers are 0.03 ns, 0.12 and 0.16 ns, respectively. The elongated lifetime with spacer from one bilayer to four bilayers should be ascribed to the decreased coupling effect between PFVP and Ag NPs with increased distance. It should be noted that when PFVP is very close to Ag NPs, energy transfer from

Figure 5. Emission spectra of the same amount of PFVP on Ag NP platforms with different extinction intensities (0.22, 0.40, 0.61) and the reference glass slide upon excitation at 430 nm. The distance between PFVP and Ag NPs is two bilayers of PDDA/PSS.

PFVP to Ag NPs could occur resulting in quenching the polymer emission with decreased fluorescence lifetime.30,46 The shortest lifetime of 30 ps was observed for PFVP deposited on Ag NP platform with one bilayer spacer. This should be due to the superposition of both plasmon effect and energy transfer. The competition between plasmon enhancement and energy transfer quenching thus resulted in a lower emission intensity for PFVP with one bilayer spacer relative to that with a two-bilayer spacer. Study of Ag NP Platform Extinction Intensities on CPE Emission. In order to study the influence of the extinction intensity of Ag NP platforms on CPE fluorescence enhancement, the PL spectra were measured for PFVP on Ag NP platforms with extinction intensities of 0.22, 0.40, and 0.61, respectively, using glass slide as a reference. The conditions for construction Ag NP-PFVP and glass-PFVP were identical to ensure that nearly equal quantity of PFVP was adsorbed onto each substrate. Here the distance between Ag NPs/glass slides and PFVP was two bilayers of (PDDA/PSS). As shown in Figure 5, the enhancement factor for PFVP on Ag NP surface is 6.5, 3.9, and 1.9 for Ag NP platform with extinction intensities of 0.61, 0.40, and 0.22, respectively. This clearly indicates that the higher Ag NP density 3285

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The Journal of Physical Chemistry B is beneficial to the enhancement of PFVP fluorescence. One logical explanation is that for the platform with elongated deposition time, more effective coupling between Ag NPs and a stronger electrical field occurs due to percolated fractal structure resulting from the shortened interparticle distance.17,28 The effective coupling between Ag NPs increases the interaction among Ag NPs and CPEs, which results in enhanced PFVP fluorescence. Effect of Electrical field on CPE Emission. To test the electrical field effect on CPE fluorescence enhancement, the emission spectra of PFVP on Ag NP platforms as well as that on reference slides upon excitation at different wavelengths were collected. The Ag NP platform has an extinction intensity of 0.61, and the distance between CPE and Ag NP platform was a twobilayer spacer of PDDA/PSS. Figure 6A shows the enhancement factor of PFVP by comparing the emission on Ag NP platform with that on reference slide at various excitation wavelengths from 300 to 450 nm. The enhancement factor changes as a function of excitation wavelength and follows the shape of the Ag NP extinction spectrum. The maximum polymer emission occurs at the excitation wavelength near the maximum of Ag NP platform extinction spectrum. Figure 6B summarizes the maximum enhancement factors observed for all four CPEs (calculated from Figure 4A and Figures S3-S5, Supporting Information). Interestingly, there is a strong correlation between the fluorescence enhancement and the degree of spectral overlap between the CPE excitation spectra and the extinction spectrum of Ag NP platform. When CPEs are placed near Ag NPs, there is often a strong absorption effect caused by the localized enhanced electromagnetic field from the incident. Ag NPs are able to modify the absorption in ways which increase the photonic mode density and incident electric field felt by the fluorophore.23,29 In addition, finite difference time-domain (FDTD) theoretical calculations also show that the magnitude of the surrounding electric field varies at different locations around metal nanoparticles.47 When the absorption spectrum of CPE has good overlap with the extinction spectrum of Ag NP platform, the enhanced electric field effect reaches the maximum, resulting in the highest fluorescence enhancement. The combination of shortened lifetime of PFVP and the excitation wavelength dependent enhancement indicate that both enhanced electrical field and plasmon-coupling could contribute to CPE fluorescence enhancement, which is

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similar to those reported for small organic dyes, quantum dots, and other fluorophores.20,47,48 ssDNA Detection on PFVP-Ag NP Platform. After discussion of the CPE fluorescence enhancement by Ag NP platforms, we examine the ability of the amplified CPE fluorescence signatures on ssDNA detection based on energy transfer. PFVP and Cy5 were selected as the donor-acceptor pair in this study due to their good spectral overlap (Figure S6 in the Supporting Information), which should favor energy transfer between them. In addition, at the Cy5 emission maximum, the emission tail of PFVP is close to zero, which indicates that there is almost no cross-talk for the donor-acceptor pair. The illustration of the detection scheme is shown in Figure 7A. The approach utilizes PFVP to enhance the signals of duplex formed between Cy5labeled PNA and ssDNA. If the ssDNA is complementary to the Cy5-PNA, the negatively charged Cy5-PNA/ssDNAc duplex would bind to the surface of positively charged PFVP-Ag NP platform. The close proximity between PFVP and Cy5 will allow FRET sensitization of Cy5 upon PFVP excitation. For noncomplementary ssDNA, no Cy5 signal can be obtained as there is no duplex formation between ssDNAnc and the neutral Cy5-PNA. Therefore, we can determine whether the solution contains the target ssDNA sequence by monitoring Cy5 emission upon PFVP excitation. As shown in Figure S7, Cy5 emission at ∼670 nm is only observed for ssDNAc, but not for ssDNAnc, which indicates good selectivity of the assay. Figure 7B shows the fluorescence intensity of Cy5-ssDNA deposited atop the surface of PFVP-AgNP platform and control glass slide, respectively. For the control slide, upon excitation at 430 nm, the polymer sensitized Cy5 emission intensity (red in Figure 7B) is ∼2-fold higher than that upon direct excitation of Cy5 at 645 nm (cyan in Figure 7B), indicative of Cy5 signal amplification provided by PFVP. Under the same experimental conditions, the polymer sensitized Cy5 emission from Ag NP platforms (black in Figure 7B) is approximately 4-fold higher than that from the reference slide (red in Figure 7B), On the other hand, upon direct excitation at 645 nm, there is about 2-fold enhancement in Cy5 emission atop the surface of Ag NPs (blue in Figure 7B) compared with that on the control glass slide (cyan in Figure 7B), which should be attributed to the direct enhancement of Cy5 by Ag NPs.33 As such, the polymer

Figure 6. (A) Fluorescence enhancement factors (EF) at various excitation wavelengths (red dots) and the extinction spectrum of Ag NP platform (black circles). (B) Fluorescence enhancement factors vs excitation wavelengths for different CPEs on Ag NP platform: (1) PFP, (2) PFVP, (3) PFBT, and (4) PFVBT. 3286

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Figure 7. (A) Schematic illustration of ssDNA detection on the surface of Ag NP-PFVP platform: (1) hybridization; (2) self-assembly. (B) PL spectra of the self-assembled reference glass slide under excitation at 430 nm (red) and 645 nm (cyan) and Ag NP platforms under excitation at 430 nm (black) and 645 nm (blue). The same amount of Cy5-ssDNA was assembled onto each slide.

sensitized Cy5 emission on Ag NP platform is 4-fold better than that upon direct excitation of the Cy5 on the same platform, which clearly demonstrates that the enhanced polymer fluorescence by Ag NPs could be successfully transformed to improve the signal output of Cy5.

’ CONCLUSION We report the fluorescence enhancement of PFVP on Ag NP platforms under different conditions. Up to 6.5-fold fluorescence enhancement is observed for PFVP at a distance of ∼6 nm from Ag NP platform with an extinction intensity of 0.61. Fluorescence decay measurements show that there is an increase in the radiative decay rates of PFVP on the Ag NP platform as compared to that on reference glass slides, indicating that plasmon effect contributes to PFVP fluorescence enhancement. The dependence of fluorescence enhancement factor on excitation wavelength and spectral overlap illustrates that an enhanced electric field component also contributes to the CPE fluorescence enhancement. The enhanced PFVP fluorescence signature by Ag NPs has been further utilized for ssDNA detection on Ag NP surface, which shows an increased signal output of Cy5 as compared to that on the reference slide. Further tuning of metal structures with suitable extinction spectra to match the excitation spectrum of each CPE could lead to enhanced optical response for different CPEs in various applications. ’ ASSOCIATED CONTENT

bS

Supporting Information. Spectroscopy study of polymer fluorescence in the absence and presence of Ag nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT The authors are grateful to the National University of Singapore (NUS ARF R-279-000-234-101) and the Singapore

Ministry of Education (R-279-000-255-112) for financial support. The authors thank K. Y. Pu and R. Y. Zhan for providing polymer samples and Dr. Q. B. Zhang and Prof. J. Y. Lee for discussion on nanoparticle synthesis.

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